The growing field of genetic therapy requires methodology for delivering nucleic acids, synthetic or natural, in the form of DNA, RNA or oligonucleotides containing modified nucleotides, to cells of an organism. The method of delivery can be diverse, and includes use of viruses, direct injection of "naked" DNA into the organism, ex vivo introduction of nucleic acids to cells by chemical-mediated transformation of the cells or electroporation, or use of liposomes.
Non-viral vectors such as liposomes recently have attracted attention as possible vehicles for nucleic acid delivery in gene therapy. In relation to viral vectors, liposomes are safer, have higher capacity, are less toxic, and are non-immunogenic. Felgner, P. L. and Ringold, G. M., Nature 337, 387-388 (1989). Among these vectors, cationic liposomes are the most studied, due to their effectiveness in mediating mammalian cell transfection in vitro. The technique, known as lipofection, employs a liposome made of nucleic acid/cationic lipid and facilitate transfection into cells. The lipid-nucleic acid complex fuses or otherwise disrupts the plasma or endosomal membranes and efficiently transfers the nucleic acid into cells. Lipofection is five to one hundred times more efficient in introducing DNA into cells than calcium phosphate or DEAE-dextran transfection methods. Chang et al., Focus 10: 66 (1988).
Cationic liposome preparations can be made by conventional methodologies. See, for example, Felgner et al., Proc. Nat'l Acad. Sci USA 84:7413 (1987); Schreier, J. of Liposome Res. 2:145 (1992); Chang et al. (1988), supra. Commercial preparations, such as Lipofectin.RTM. (Life Technologies, Inc., Gaithersburg, Md. USA), also are available. The amount of liposomes and the amount of DNA can be optimized for each cell type based on a dose response curve. Felgner and Ringold (1989), supra.
Cationic lipids are not found in nature, and DNA/cationic liposomes complexes remain quite cytotoxic, as these complexes appear incompatible with the physiological environment in vivo which is rich in anionic molecules. They may also have undesirable non-specific interactions with negatively charged serum components, blood cells, and the extracellular matrix in vivo.
Another serious limitation in the use of DNA/cationic liposome complexes has been the lack of success in targeting these complexes by use of tissue-specific ligands. This is probably due to the presence of cationic charge in the complex which leads to excessive non-specific tissue uptake.
When cationic liposomes are used as DNA vectors, DNA is complexed with the liposomes via charge interaction. Since DNA usually cannot be optimally condensed by cationic liposomes, the DNA/liposome complex is unstable, i.e., it undergoes slow aggregation. The aggregation increase at high DNA concentration, which is required for clinical applications. Therefore, DNA/cationic liposome complexes need to be prepared fresh, which reflects in increased cost and decreased convenience.
Anionic liposomal vectors have also been examined. These include pH sensitive liposomes which disrupt or fuse with the endosomal membrane following endocytosis and endosome acidification. With conventional technology, DNA encapsulation with anionic liposomes requires very high lipid concentration and entrapment efficiency rarely exceeds 20%. Only a small portion of the liposomes generated contains DNA. The poor DNA encapsulation efficiency achieved by conventional methods is due in large part to the relatively large size of uncondensed DNA and its inability to interact with the anionic liposomes. Procedures commonly used to increase liposomal entrapment efficiency, such as repeated freeze-thaw cycles and polycarbonate membrane extrusion can also lead to severe damage to the DNA strands and loss of DNA expression activity. Zhou, X., Klibanov, A. L., and Huang, L., J. Lip. Res. 2: 125-39 (1992).
Other non-liposomal lipidic vectors, both cationic and anionic, such as oil-in-water emulsions and micelles composed of various anionic, non-ionic or cationic surfactants or surfactant mixtures, also potentially can complex with DNA and mediate DNA delivery. These vectors share many of the same problems associated with liposomal vectors, such as non-specific uptake, cytotoxicity and low encapsulation efficiency.
DNA/liposome complexes are sometimes targeted to the cell type or tissue of interest by the addition to the liposome preparation of a ligand, usually a polypeptide, for which a corresponding cellular receptor has been identified. An example of a cell receptor that can be targeted is the folate receptor which has recently been identified as a prominent tumor marker, especially in ovarian carcinomas. KB cells are known to vastly overexpress the folate receptor. Campbell et al., Cancer Res. 51: 6125-6132 (1991). Yet other targeting ligands have been examined for liposome targeting including transferrin, protein A, ApoE, P-glycoprotein, .alpha..sub.2 -macroglobin, insulin, asiolofetuin, asialoorosomucoid, monoclonal antibodies with a variety of tissue specificity, biotin, galactose or lactose containing haptens (monovalent and tri-antennary), mannose, dinitrophenol, and vitamin B12. The ligands are covalently conjugated to a lipid anchor in either pre-formed liposomes or are incorporated during liposome preparation. Lee and Low J. Biol. Chem. 269: 3198-3204 (1994) and Lee and Low Biochim. Biophys. Acta 1233: 134-144 (1995).
Synthetic peptides could be incorporated into DNA/liposome complexes to enhance their activity, or to target them to the nucleus. For example, in order to gain access to the cytoplasm, the DNA molecule must overcome the plasma membrane barrier. In nature, viral fusion peptides facilitate the delivery of viral DNA into the cytoplasm by promoting viral membrane fusion with the plasma membrane. For recent reviews on this subject see Stegmann et al., Ann. Rev. Biophys. Chem. 18: 187-221 (1989). For the influenza virus, the hemagglutinin (trimer) HA peptide N-terminal segment (a hydrophobic helical sequence) is exposed due to a conformational change induced by acidic pH in the endosomes (pH 5-6), inserts into the target membrane, and mediates the fusion between the virus and the target endosomal membrane. Weber et al., J. Biol. Chem. 269: 18353-58 (1994). Recently, several amphipathic helix-forming oligopeptides have been designed to imitate the behavior of the viral fusion peptide. See, for example, Haensler and Szoka, Bioconj. Chem. 4: 372-79 (1993). Existing technology for use of these peptides require either the covalent conjugation of the peptide to a DNA-binding molecule such as a polycation (for an example see Haensler and Szoka, supra, or the inclusion in the transfection medium of a high concentration of the peptides. The disadvantage of covalent conjugation of peptides to a polycation is that the formation of a high molecular weight conjugate is known to invoke an immune response (including T-cell and antibody responses) against the peptide in vivo. While free oligopeptides are generally non-immunogenic, maintaining a high peptide concentration in the medium is prohibitively expensive and incompatible with in vivo gene therapy.
Expression of DNA into a protein product in mammalian cells requires that transcription of DNA into mRNA takes place in the nucleus. Therefore, the DNA moiety in the liposome of the invention needs to cross the nuclear envelope for the expression of said DNA inside a cell. Nuclear localization signal peptides, when attached covalently to a macromolecule such as a protein, have been shown to facilitate their translocation into the nucleus. Goldfarb et al., Nature 322: 641-44 (1986); Shreiber et al., Med. Sci. 8: 134-39 (1992). It has been proposed that this could also be used to facilitate delivery of liposome contents. Again, the disadvantage of covalent conjugation of peptides to a polycation is that the formation of a high molecular weight conjugate is known to invoke an immune response, including T-cell and antibody responses, against the peptide in vivo.